US5792744A - Proteins from mammalian liver - Google Patents

Proteins from mammalian liver Download PDF

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Publication number
US5792744A
US5792744A US08/765,957 US76595797A US5792744A US 5792744 A US5792744 A US 5792744A US 76595797 A US76595797 A US 76595797A US 5792744 A US5792744 A US 5792744A
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United States
Prior art keywords
proteins
protein
liver
extracted
antibodies
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Expired - Fee Related
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US08/765,957
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English (en)
Inventor
Severino Ronchi
Alberto Bartorelli
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Zetesis SpA
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Zetesis SpA
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Assigned to ZETESIS, S.P.A. reassignment ZETESIS, S.P.A. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BARTORELLI, ALBERTO, RONCHI, SEVERINO
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Classifications

    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F01—MACHINES OR ENGINES IN GENERAL; ENGINE PLANTS IN GENERAL; STEAM ENGINES
    • F01D—NON-POSITIVE DISPLACEMENT MACHINES OR ENGINES, e.g. STEAM TURBINES
    • F01D25/00—Component parts, details, or accessories, not provided for in, or of interest apart from, other groups
    • F01D25/28—Supporting or mounting arrangements, e.g. for turbine casing
    • F01D25/285—Temporary support structures, e.g. for testing, assembling, installing, repairing; Assembly methods using such structures
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F23—COMBUSTION APPARATUS; COMBUSTION PROCESSES
    • F23R—GENERATING COMBUSTION PRODUCTS OF HIGH PRESSURE OR HIGH VELOCITY, e.g. GAS-TURBINE COMBUSTION CHAMBERS
    • F23R3/00—Continuous combustion chambers using liquid or gaseous fuel
    • F23R3/42—Continuous combustion chambers using liquid or gaseous fuel characterised by the arrangement or form of the flame tubes or combustion chambers
    • F23R3/60—Support structures; Attaching or mounting means
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B23—MACHINE TOOLS; METAL-WORKING NOT OTHERWISE PROVIDED FOR
    • B23P—METAL-WORKING NOT OTHERWISE PROVIDED FOR; COMBINED OPERATIONS; UNIVERSAL MACHINE TOOLS
    • B23P2700/00—Indexing scheme relating to the articles being treated, e.g. manufactured, repaired, assembled, connected or other operations covered in the subgroups
    • B23P2700/13—Parts of turbine combustion chambers

Definitions

  • the present invention refers to proteins from animal tissues, particularly from mammalian liver, and to the use thereof in oncology.
  • WO 92/10197 discloses extracts of mammalian organs, particularly of goat liver, consisting of at least three different proteins and characterized by unusual pharmacological and immunological properties. No information was reported on the actual role and on the sequences of the individual protein components.
  • a 23-KDa dimeric protein extracted with 5% perchloric acid from rat liver and kidney has been disclosed in Eur. J. Biochem. 212, 665, 1993.
  • the corresponding cDNA sequence have been deposited at the EMBL data bank under accession number X70825.
  • HMG High-mobility group
  • WO 93/18146 discloses a protein extracted from rabbit-liver having a molecular weight of 59 Kd capable of complexing with chaperons and a heat shock protein of 90 Kd.
  • Said protein is useful in oncology in view of the following properties:
  • the serum of animals immunized with the protein displays cytotoxic activity against human tumor cell cultures
  • the protein has marked antineoplastic activity at the dose of 0.015 ⁇ g/kg in Balb/c mice having a murine colon adenocarcinoma (c26) and in rats with intrapleuric Yoshida ascitic tumor;
  • the protein of the invention has a high degree of homology with that extracted from rat liver disclosed in Eur. J. Biochem. 212, 665, 1993.
  • Proteins having a high degree of homology with that of Sequence Id No. 1 have also been found in liver of different animal species, particularly bovine and equine liver.
  • a new protein family has been therefore found: the members of this previously unknown family are characterized by an high degree of conservation and homology between the mammalian species and a molecular weight ranging from about 10 KDa to about 14 KDa.
  • high degree of homology means an homology of the aminoacid sequences of about 80% or higher, preferably of 90% or higher.
  • the invention further refers to the use in oncology, as a therapeutic and/or diagnostic tool, of the above mentioned perchloric acid extractable proteins from mammalian liver.
  • compositions of the invention will be administered by parenteral route, preferably subcutaneously or intra-muscularly and will typically contain from 0.1 to 50 mg of total protein per unit dose.
  • the protein active principle purified by conventional methods, may be lyophilized on a suitable non-toxic carrier and distributed in vials or bottles.
  • Suitable solvents include sterile water or saline solutions.
  • the proteins of the invention or fragments thereof, produced for instance by chemical synthesis may be used to produce polyclonal or monoclonal antibodies.
  • Particularly interesting antibodies recognize tumoral antigens and are therefore useful for diagnostic, therapeutic or research purposes. Two of said antibodies have been deposited on 27 Jul., 1993 at the European collection of Animal Cell Cultures (EGACC), Porton Down, Salisbury, UK under accession numbers 930806103 and 930806104.
  • the proteins of the invention when administered to patients affected by neoplastic disease, in addition to advantageous effects such as inhibition or regression of the tumoral mass, reduction of pain and improvement of cenestesis, raise antibodies having marked cytotoxic action on cultured tumor cells.
  • the whole serum, not free from the complement cascade, is required for said cytotoxic effect.
  • the proteins of the invention When used for therapeutic purposes or as a vaccine to induce immunity against neoplastic transformation, the proteins of the invention may be administered at a dosage ranging from 0.1 to 30 mg/day/patient, by the subcutaneous, intramuscular or intravenous route. The treatment will be repeated even for long periods, until the concentration of the raised antibodies reaches a convenient level.
  • the concentration of the raised antibodies may be determined by conventional methods, using for instance immunoenzymatic techniques.
  • the invention provides diagnostic kits containing suitably labelled reagents, e.g. the protein of the invention or fragments thereof as an antigen, optionally immobilized on a suitable support, anti-Ig antibodies and suitable reagents able to detect, e.g. by means of a colorimetric reaction, an antigen-antibody complex.
  • the proteins of the invention may advantageously be administered together with suitable carriers, acting as adjuvants.
  • suitable adjuvants may be selected, for instance, from non-toxic proteins, preferably xenogenic proteins, e.g. proteins from the same species from which the immunogenic protein is extracted.
  • the proteins of the invention are prepared by subjecting the crude extract, obtained by extracting the organs with perchloric acid and subsequently with hypertonic saline solutions (KCl 3M for instance) and subsequent dialysis, to purification steps in HPLC and hydrophobic exchange chromatography (FPLC) as hereinafter specified in the Examples.
  • the protein obtained from goat liver is blocked at the N-terminal and it has been therefore partially sequenced after cleavage with CNBr, yielding two main fragments having molecular weight (determined by the MALDI-TOF method) respectively of 10263 and 4063 D, respectively, whereas the molecular weight before cleavage is 14.290 Daltons, in agreement with the value determined by SDS-PAGE electrophoresis.
  • a liver goat extract prepared as in WO 92/10197, and hereinafter referred to as UK 101, is concentrated on Amicon PM 10 membrane and subsequently dialyzed against NaH 2 PO 4 /Na 2 HPO 4 , 0.01M, pH 6.5.
  • the product is purified by HPLC on TSK DEAE 5 PW equilibrated in said buffer; the starting buffer is collected and the protein absorbed on the resin are eluted with 1M NaCl.
  • the peak eluted in the starting buffer is subsequently purified by HPLC on TSK SW 3000 column.
  • the purification on this hydrophobic exchange column is carried out in the following conditions: a starting buffer, Tris HCl 20 mM pH 7 containing (NH 4 ) 2 SO 4 1M, is first eluted, followed by a linear gradient elution ending with Tris- HCl 20 mM without ammonium sulfate. The starting buffer is discarded whereas the zone, eluted in the gradient at a (NH 4 ) 2 SO 4 molarity ranging from 0.6 to 0.8M is collected and dialyzed against H 2 O.
  • UK 114 A sample hereinafter referred to as UK 114 showing a protein band in SDS-PAGE of about 14 Kda with a purity degree of about 90% is obtained.
  • Monoclonal antibodies were obtained from Balb/c mice one month after weekly subcutaneous injections of 100 ⁇ g of UK 101 with incomplete Freund's adjuvant.
  • the fusion with myeloma cells of lymphocytes obtained from animals immunized against UK 101 was carried out by conventional methods. Two of the obtained hybridomas were deposited on 27 Jul., 1993 at the European Collection of Animal Cell Cultures (ECACC) Porton Down, Salisbury, UK, under accession numbers 930806103 and 930806104.
  • the antibodies secreted by said hybridome recognize the proteins of the invention.
  • the mono- and polyclonal antibodies have been assayed in immunocytochemistry tests on 30 bioptic samples of malignant tumors isolated from different organs such as breast, lung, bladder, stomach, colon-rectum, uterus, soft tissues, prostate.
  • the tissues were fixed in 10%, buffered formaline and preparations in paraffine were stained by means of Mistostain Kit SP, Zymed Lab. Inc.
  • the sections were incubated with the antibodies (0.5 ⁇ g/ml of Ig with 1% BSA/PBS) overnight at 4° C. After washing, the slides were incubated with anti-rabbit pig biotinylated Ig for 60 minutes and then for other 60 minutes with a 1:100 dilution of peroxidated streptavidine-biotine complex. The peroxidase binding was detected using the 3,3-diaminobenzidine/H 2 O 2 reaction. Only the tissue showing specific reaction against the antibodies in the cytoplasma were considered positive. The immunoreactivity was considered as negative, slightly positive, positive (++) and highly positive (+++) for the normal tissues. The results are reported in the following Table.
  • the immunocytochemical reactivity with different polyclonal antibodies anti goat, calf and horse liver extract is detectable in most malignant tumors (82.7% for antibodies against horse liver extract and 100% for calf liver extract).
  • the monoclonal antibody secreted by the hybridoma No. 930806103 gave positive results for 93.7% of the assayed tumors.

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  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Mechanical Engineering (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Combustion & Propulsion (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
US08/765,957 1994-07-14 1995-07-12 Proteins from mammalian liver Expired - Fee Related US5792744A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
ITMI94A001469 1994-07-14
ITMI941469A IT1270618B (it) 1994-07-14 1994-07-14 Proteine ad attivita' antitumorale
PCT/EP1995/002723 WO1996002567A1 (en) 1994-07-14 1995-07-12 Proteins from mammalian liver and their use in oncology

Publications (1)

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US5792744A true US5792744A (en) 1998-08-11

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US08/765,957 Expired - Fee Related US5792744A (en) 1994-07-14 1995-07-12 Proteins from mammalian liver

Country Status (20)

Country Link
US (1) US5792744A (hu)
EP (1) EP0770093A1 (hu)
JP (1) JPH10502814A (hu)
KR (1) KR100425627B1 (hu)
CN (1) CN1146576C (hu)
AU (1) AU702294B2 (hu)
BR (1) BR9508382A (hu)
CA (1) CA2194861A1 (hu)
CZ (1) CZ289380B6 (hu)
FI (1) FI970097A (hu)
HU (1) HU218285B (hu)
IL (1) IL114561A (hu)
IT (1) IT1270618B (hu)
MX (1) MX9602557A (hu)
NO (1) NO970114L (hu)
RU (1) RU2163243C2 (hu)
TR (1) TR199500854A2 (hu)
TW (1) TW434257B (hu)
WO (1) WO1996002567A1 (hu)
ZA (1) ZA955837B (hu)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001017535A2 (en) * 1999-09-03 2001-03-15 Vigen Laboratories, Inc. Enriched fraction from a porcine liver extract for treating human diseases
US6207200B1 (en) * 1996-09-13 2001-03-27 Zetesis S.P.A. Use of proteins as anti-retroviral agents
WO2002083161A1 (en) * 2001-04-10 2002-10-24 Zetesis S.P.A. Use of the protein uk114 or of fragments thereof for the treatment and prevention of the endotoxic shock

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1276707B1 (it) * 1995-06-13 1997-11-03 Zetesis Spa Composizioni farmaceutiche ad attivita' analgesica
IT1282608B1 (it) 1996-02-13 1998-03-31 Zetesis Spa Sequenza oligonocleotidica da fegato di capra
CA2266346A1 (en) * 1996-09-18 1998-03-26 Zetesis S.P.A. Use of proteins as agents against autoimmune diseases
IT1290828B1 (it) * 1997-03-25 1998-12-11 Zetesis Spa Uso di proteine estraibili da organi animali per la preparazione di medicamenti per il trattamento di condizioni patologiche
IT1298442B1 (it) * 1998-02-24 2000-01-10 Zetesis Spa Composizioni orali a basso dosaggio di proteine citotossiche
US20010014471A1 (en) * 1999-04-15 2001-08-16 Vytautas Naktinis Recombinant protein and its use in therapy and diagnostics
ITMI20022307A1 (it) * 2002-10-30 2004-04-30 Zetesis Spa Associazioni anti-tumorali comprendenti proteine e chemioterapici.
CN104610762A (zh) * 2015-01-16 2015-05-13 青岛新诺科铸造材料科技有限公司 一种填充型精密铸造低温调制蜡及其制备方法
IT201600127428A1 (it) * 2016-12-16 2018-06-16 Cusani Alberto Bartorelli Nuova proteina ricombinante uk 114 in forma stabile polimerica per uso nella terapia, nella diagnostica e nella prevenzione di neoplasie maligne
IT201900022203A1 (it) 2019-11-26 2021-05-26 Cusani Alberto Bartorelli Proteine uk 114 da salmone per uso nella terapia, nella diagnostica e nella prevenzione di neoplasie maligne

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010197A1 (en) * 1990-12-11 1992-06-25 Zetesis S.P.A. Substances of polypeptide nature useful in human therapy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992010197A1 (en) * 1990-12-11 1992-06-25 Zetesis S.P.A. Substances of polypeptide nature useful in human therapy
EP0574394A1 (en) * 1990-12-11 1993-12-22 Zetesis Spa SUBSTANCES OF PEPTIDIC NATURE USEFUL IN HUMAN THERAPY.

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Database EMBL, Emest:Hs68065, Access no: T98680, The WashU merck EST Project , L. Hillier et al, 17 Apr. 1995, See Abstract. *
Database EMBL, Emest:Hs68065, Access-no: T98680, "The WashU-merck EST Project", L. Hillier et al, 17 Apr. 1995, See Abstract.
Database EMBL, Emrod:Respspl; Access no: D49363, Sequence of PSP1 , T. Oka, 22, Feb. 1995, See Abstract. *
Database EMBL, Emrod:Respspl; Access-no: D49363, "Sequence of PSP1", T. Oka, 22, Feb. 1995, See Abstract.
Eur. J. Biochem, (1993), 212(3), 665 73, Characterization, purification and cDNA cloning of a rat perchloric acid soluble 23 kDa protein present only in liver and kidney , Levy Favatier et al. *
Eur. J. Biochem, (1993), 212(3), 665-73, "Characterization, purification and cDNA cloning of a rat perchloric-acid-soluble 23-kDa protein present only in liver and kidney", Levy-Favatier et al.
Lewin (Science, 1988, 237:1570). *
Reeck et al (Cell, 1987, 50:667). *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6207200B1 (en) * 1996-09-13 2001-03-27 Zetesis S.P.A. Use of proteins as anti-retroviral agents
WO2001017535A2 (en) * 1999-09-03 2001-03-15 Vigen Laboratories, Inc. Enriched fraction from a porcine liver extract for treating human diseases
WO2001017535A3 (en) * 1999-09-03 2001-07-26 Vigen Lab Inc Enriched fraction from a porcine liver extract for treating human diseases
WO2002083161A1 (en) * 2001-04-10 2002-10-24 Zetesis S.P.A. Use of the protein uk114 or of fragments thereof for the treatment and prevention of the endotoxic shock
US20040180835A1 (en) * 2001-04-10 2004-09-16 Alberto Bartorelli Use of the protein uk114 or of fragments thereof for the treatment and prevention of the endotoxic shock

Also Published As

Publication number Publication date
TR199500854A2 (tr) 1996-06-21
EP0770093A1 (en) 1997-05-02
FI970097A (fi) 1997-03-06
ZA955837B (en) 1996-02-21
FI970097A0 (fi) 1997-01-10
CZ289380B6 (cs) 2002-01-16
TW434257B (en) 2001-05-16
CN1152924A (zh) 1997-06-25
ITMI941469A1 (it) 1996-01-14
IT1270618B (it) 1997-05-07
HUT76328A (en) 1997-08-28
HU9700057D0 (en) 1997-02-28
KR100425627B1 (ko) 2004-06-18
CN1146576C (zh) 2004-04-21
IL114561A (en) 2000-11-21
JPH10502814A (ja) 1998-03-17
HU218285B (en) 2000-07-28
AU3077995A (en) 1996-02-16
ITMI941469A0 (it) 1994-07-14
CZ6997A3 (en) 1997-08-13
RU2163243C2 (ru) 2001-02-20
AU702294B2 (en) 1999-02-18
NO970114L (no) 1997-03-05
NO970114D0 (no) 1997-01-10
IL114561A0 (en) 1995-11-27
BR9508382A (pt) 1997-12-23
MX9602557A (es) 1997-05-31
CA2194861A1 (en) 1996-02-01
WO1996002567A1 (en) 1996-02-01

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